This application focuses on the mechanism by which thrombin, a serine protease formed upon completion of the coagulation cascade, induces a myriad of responses in the endothelial cell. Thrombin has been recently shown to activate several members of the protease activated receptor (PAR) family. In the endothelial cell, these interactions lead to a variety of responses including proliferation, expression of leukocyte adhesion molecules, and increased permeability. The PAR receptors are known to be coupled to G proteins, but the specific isoforms of the Galpha subunit which mediate endothelial cell responses are not yet known. The goal of this application is to understand the molecular basis of receptor-G protein interactions by studying the C-terminal regions of the Galpha subunits that interact with the cytoplasmic tail of coupled receptors. The specific aims are to [1] identify G proteins activated by PARs in endothelial cells and determine which thrombin-mediated signaling events are mediated by which G proteins, [2] identify mechanisms of interaction between PARs and G proteins using a combinatorial library of C-terminal G protein peptides, [3] determine the functional effects of blocking peptides introduced through minigene transfection, and [4] determine the specificity of peptide antagonists that block endothelial cell activation by thrombin receptors or other agonists such as bradykinin and histamine. The PI proposes a number of creative and imaginative techniques to address these aims.